This project will explore a direct role for HCMV in TA, by focusing on quantitation of viral DNA and mRNA in peripheral blood and endomyocardial biopsies as well as by studying virus-encoded and -induced chemokines and cytokines in cultured endothelial and smooth muscle cells with the expectation of revealing correlates of disease development and progression. The 1st specific aim will evaluate peripheral blood granulocyte and mononuclear leukocyte populations as well as endomyocardial biopsies for levels of viral DNA and viral gene expression. Solution PCR and RT-PCR methods, as well as in situ antibody staining and hybridization will be used to establish the levels and tissue or cell distribution of viral infection in patients. This study design should differentiate latent from productive infection. The 2nd specific aim will investigate the role of virus-encoded and -induced chemokines as determinants of host cell trafficking that facilitate inflammatory cell migration, possibly contributing to the development of TA. The HCMV chemokine (UL146/vCXC-l), a powerful proinflammatory signal and one of the most highly variable genes known (comparing virus strains), is predicted to control strain differences in promoting migration of different inflammatory cell types or levels. The activity of vCXC-1 from strains of virus associated with TA will be compared to standard strains in ability to attract neutrophils or mononuclear cell types via a specific interaction with the receptor hCXCR2 as well as through other as yet undescribed receptors. The ability of HCMV infection to induce a restricted set of host cytokines (IL-6 and IL-1) as well as chemokines (MCP-1 and IL-8) will be extended such that human cDNA microarrays representing over 30,000 genes will be surveyed, with a focus on the behavior of strains from transplant recipients in aortic endothelial cell cultures. Thus, possible reasons underlying progression to TA that result from proinflammatory (or other vascular) processes controlled by viral effects on infected cells will be defined. These results will be correlated with the expression of viral and host chemokines in cardiac biopsy specimens prior to and during the progression of TA. The 3rd specific aim of this project will investigate signaling and cell migration controlled by HCMV US28 chemokine receptor, a receptor that has been implicated in smooth muscle cell migration during atherosclerosis. Here we will evaluate the four possible roles of the US28 receptor as a contributor to TA: smooth muscle cell migration, smooth muscle or endothelial cell activation, infected cell adhesion to endothelial cells and immunomodulation through function as a chemokine sink. The overall goals of the project could greatly increase the understanding of the direct impact of HCMV on TA and could lead to viral and host diagnostic indicators predicting progression to TA.